Silencing of IRF8 Mediated by m6A Modification Promotes the Progression of T‐Cell Acute Lymphoblastic Leukemia

Abstract T‐cell acute lymphoblastic leukemia (T‐ALL) is an aggressive hematological malignancy with a poor prognosis, urging for novel therapeutic targets and treatment strategies. N6‐methyladenosine (m6A) is a crucial methylation modification that affects the pathogenesis of leukemia by regulating the mRNA of key genes. Interferon regulatory factor 8 (IRF8) is a crucial transcription factor for hematological lineage commitment, but its role in T‐ALL is unclear. Here, IRF8 is shown to suppress T‐ALL. The expression of IRF8 is abnormally silenced in patients with T‐ALL. Knockout of Irf8 significantly hastens the progression of Notch1‐induced T‐ALL in vivo. Overexpression of IRF8 suppresses the proliferation and invasion of T‐ALL cells by inhibiting the phosphatidylinositol 3‐kinase/AKT signaling pathway. The fat mass‐ and obesity‐associated protein (FTO), an m6A demethylase, is responsible for directly binding to m6A sites in 3′ untranslated region of IRF8 messenger RNA (mRNA) and inducing mRNA degradation via m6A modification. Targeting the FTO‐IRF8 axis is used as a proof of concept therapy; inhibition of FTO's demethylase activity drastically alleviates the proliferation of leukemic cells and prolongs the survival of T‐ALL mice by restoring IRF8 expression. This study elucidates the pathogenesis of T‐ALL from the perspective of epitranscriptomics and provides new insight into the genetic mechanisms and targeted therapy of T‐ALL.


Migration and invasion assay
The migration potential was measured using transwell inserts fitted with 8 μm-pore-size polycarbonate filters (Corning Costar, USA) in a 24 well plate. Cells were seeded in the upper compartment in 0.5% FBS culture medium while the medium in the lower compartment contained 20% FBS. 24 h later, contents in the lower compartments were harvested and counted. The ratio of migrated cells to the total number of cells seeded in the upper compartments was considered as the migration rate.
Furtherly, the invasive potential was measured using transwell inserts coated with Matrigel (Corning Costar, USA) on the upper compartment. Cells were seeded in the upper compartment in 0.5% FBS medium while the culture medium in the lower compartment contained 20% FBS. 48 h later, the remaining cells in the upper compartment were gently pipetted out. At the same time, the cells on the underside of the membrane that had penetrated the filter were 4% paraformaldehyde-fixed and stained with 0.2% crystal violet. Finally, the cells were observed and captured on an inverted microscope. Besides, the migrated cells in the lower chamber were also collected by centrifugation and counted by cell counter. The ratio of migrated cell number to the total number of cells seeded was considered as the invasion rate.

Real-time quantitative-polymerase chain reaction (qRT-PCR)
Total RNA was isolated by using TRIzol (Invitrogen). For mRNA expression, 500 ng

Immunohistochemistry
For the immunohistochemical study, heat-induced epitope retrieval was applied for tissue sections before incubation with anti-IRF8, anti-PIK3R5, anti-Ki67 (Proteintech, USA), and anti-p-AKT (CST, USA) antibodies at 4℃ overnight. Then the sections were incubated with appropriate secondary antibodies conjugated to biotin-streptavidin horseradish peroxidase (HRP) for 30 min at room temperature and stained using 3,3′ -diaminobenzidine (DAB) reagent. The slides were observed and photographed under a fluorescence microscope (Invitrogen, USA), and immunostaining results were analyzed using ImageJ software.

RNA-sequence
Total RNA was extracted from IRF8 overexpressed Molt4 cells or control Molt4 cells, as well as FB23-2 treated or control Molt4 cells using TransZol Up Plus RNA Kit (Cat#ER501-01, Trans). After that, qualified total RNA was purified using RNase-Free DNase Set (QIAGEN, Germany), and RNAClean XP Kit (Beckman Coulter, USA) following the manufacturer's instructions. VAHTS Universal V6 RNA-seq Library Prep Kit (Vazyme, China) was used to prepare the libraries.
Illumina NovaSeq6000 was used to conduct sequence in each group in triplicate (Biotechnology Corporation, China). Sequence reads were aligned to the human genome version 38 (hg38) following the standard analysis pipeline for the Illumina sequence.

Dot blot analysis
Total RNA was extracted with TRIzol (Invitrogen) and quantified by spectrophotometry (Denovix). Then 2 μL RNA sample was dropped on the front of the positively-charged Nylon transfer membrane (RPN303B, GE Healthcare) and UV crosslinked twice to the Nylon transfer membrane after dried. Later, blockage of the membrane was performed with 5% skim milk, followed by overnight incubation with the anti-m6A antibody (Synaptic Systems, Germany) at 4 °C. After 1h-incubation with the corresponding second antibody, the membrane was developed and visualized with FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences, USA). Semi-quantitative analysis of blot density was conducted using ImageJ software.

Analysis of m6A/A ratio using liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Poly(A) mRNA was isolated from FB23-2 treated Molt4 cells and control cells. 8 μL mRNA was added into an 8μL mixture containing Tris-HCL, benzonase, phosphodiesterase Ⅰ, alkaline phosphatase, and RNase-free water. The mixture solution was then incubated at 37 ℃ for 5 h. After that, the solution was diluted 6 times to obtain the gradient concentration, and 10 μL of the diluted solution was loaded for measurement using Waters Acquity UPLC and AB SCIEX 5500 QQQ -MS.
For the quantification of the m6A modified nucleosides, the nucleosides to base ion mass transitions of 282.1 to 150.1 Da were accessed under the MRM positive ESI mode. Data were analyzed using MultiQuant.